Binding Between HIV-1 RT and IN and Its Functional Signification

描述(申请人提供)：逆转录病毒的RNA基因组进入细胞后，通过逆转录酶(RT)转化为cdna拷贝。对于生产性感染，产生的病毒cDNA必须整合到宿主染色体中，这一过程由整合酶(IN)催化。HIV-1IN的某些突变似乎特别地损害了逆转录，而对生命周期的其他步骤没有明显的影响。然而，人们对这种缺陷的潜在机制知之甚少。在体外，RT和IN在物理上相互作用，但这种RT-IN相互作用的生物学相关性尚不清楚。我们假设RT和IN之间的物理相互作用对于启动HIV-1复制过程中的逆转录是功能和关键的。利用核磁共振波谱，我们鉴定了IN上RT相互作用的表面，并利用基于表面等离子体共振的生物传感器测定了RT-IN复合体的亲和力和形成动力学。本应用的目的是更好地了解HIV-1RT和IN之间的物理相互作用，并确定RT-IN相互作用在HIV-1复制过程中的功能意义。其具体目的是(1)表征HIV-1IN和RT之间的物理相互作用，并证实RT和IN之间的相互作用发生在体内，以及(2)确定HIV-1RT-IN相互作用的生物学意义。在目标1中，我们将使用靶向蛋白质足迹方法定位RT的IN相互作用结构域。RT和IN在复制过程中的物理相互作用将通过使用纯化的病毒粒子和感染细胞的细胞质提取物进行免疫共沉淀实验来证实。在目标2中，我们将通过破坏假定的IN-RT结合界面和评估病毒复制来测试感染过程中RT-IN相互作用的生物学相关性。我们还将筛选能够补偿RT-非相互作用IN突变的RT突变体，并检查其他已知在反转录中产生缺陷的IN突变体，以了解它们结合RT的能力。最后，我们将使用基于荧光的高通量筛选来识别RT-IN相互作用的小分子抑制剂，并确定这些抑制剂是否也能阻止病毒复制。在这个过程中，我们将阐明两种关键的逆转录病毒酶之间的相互作用，以及这种相互作用对逆转录和病毒复制的影响。RT-IN相互作用的特征及其生物学意义的确定可能揭示IN的新功能，为某些IN突变观察到的表型提供机制基础，并为抗HIV治疗寻找新的潜在靶点。公共卫生相关性：这项提议的目的是了解人类免疫缺陷病毒I型(HIV-1)的两种关键酶--逆转录酶和整合酶之间物理相互作用的重要性。相互作用的特征及其生物学意义的确定可能揭示整合酶的新的功能作用，并提供对病毒基础生物学的更深层次的了解。此外，这项研究可能会为抗艾滋病毒治疗确定新的潜在靶点。

DESCRIPTION (provided by applicant): After cell entry, the RNA genome of retroviruses is converted to a cDNA copy by reverse transcriptase (RT). For productive infection, the resulting viral cDNA must be integrated into the host chromosome, a process catalyzed by integrase (IN). Certain mutations of HIV-1 IN appear to specifically impair reverse transcription with no apparent effects on other steps in the life cycle. However, the underlying mechanism for this defect is poorly understood. In vitro, RT and IN physically interact, but the biological relevance of this RT- IN interaction is not known. We hypothesize that the physical interaction between RT and IN is functional and critical for initiating reverse transcription during HIV-1 replication. Using nuclear magnetic resonance spectroscopy, we have identified the RT-interacting surface on IN, and have determined the affinity and kinetics of RT-IN complex formation by using a surface plasmon resonance-based biosensor. The goals of this application are to gain a better understanding of the physical interaction between HIV-1 RT and IN, and to determine the functional significance of the RT-IN interaction during HIV-1 replication. The specific aims are (1) to characterize the physical interaction between HIV-1 IN and RT and confirm that the interaction between RT and IN occurs in vivo, and (2) to determine the biological significance of the HIV-1 RT-IN interaction. In Aim 1, we will map the IN-interacting domain of RT using a targeted protein footprinting method. The physical interaction between RT and IN during replication will be confirmed by carrying out a co-immunoprecipitation experiment using purified virions and cytoplasmic extracts from infected cells. In Aim 2, we will test the biological relevance of the RT-IN interaction during infection by disrupting the putative IN-RT binding interface and assessing viral replication. We will also screen for RT mutants that can compensate for the RT- noninteracting IN mutations and examine other IN mutants known to produce defects in reverse transcription for their ability to bind RT. Finally, we will identify small-molecule inhibitors of the RT-IN interaction using a fluorescence-based high-throughput screen and determine whether such inhibitors also block viral replication. In the process, we will shed light on the interaction between two key retroviral enzymes and the effect of such an interaction on reverse transcription and viral replication. Characterization of the RT-IN interaction and determination of its biological significance may reveal new functional roles for IN, provide a mechanistic basis for the phenotypes observed with certain IN mutants, and identify new potential targets for anti-HIV therapy. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to understand the importance of the physical interaction between two key enzymes, reverse transcriptase and integrase, of human immunodeficiency virus type I (HIV-1). Characterization of the interaction and determination of its biological significance may reveal new functional roles for integrase and provide a deeper understanding of the basic biology of the virus. Furthermore, the study may identify new potential targets for anti-HIV therapy.